Blue copper proteins (BCP s) are a well-characterized family of electron transfer proteins. This proposal will focus on the synthesis of BCP's. Native ligation techniques will be employed in a segment condensation strategy to prepare BCP polypeptides (99-140 amino acids). Total chemical synthesis will allow us to introduce perturbations in BCP frameworks that are not possible through traditional mutational methods. Specifically, three uninvestigated aspects of BCP active sites will be probed: (1) Hydrogen bonding interactions between backbone amide protons and the conserved thiolate ligand are thought to be important in modulating the reduction potential of BCP's. We will investigate these effects by systematically removing these hydrogen bonds by replacing the appropriate amides with ester groups. (2) In azurin (Az), a backbone carbonyl group is thought to be the fifth donor in a roughly trigonal coordination sphere, and may play a role in regulating the reduction potential and reactivity of Az. This interaction will be removed by replacement of Gly45 with a peptide analog lacking a carbonyl group. (3) The size and composition of the loops between conserved ligand residues varies between members of the BCP family, and is thought to influence the coordination geometry at the metal. Total synthesis will allow us to modify the size of the loops between ligand residues by single atom increments and systematically investigate relationships between copper coordination geometry and reactivity. This project will allow me to acquire expertise in chemical synthetic methods and apply my background in biophysical methods while increasing the understanding of metalloprotein structure and function.